| ObjectiveAlzheimer’s disease(AD)is a neurodegenerative disease characterized by hidden onset and progressive cognitive impairment.To date,there is no disease modification therapy or cure.The drugs currently approved for the treatment of AD can only improve symptoms and cannot delay progression.The effectiveness varies from person to person.Through nearly 30 years of clinical research,our team has established a sequential treatment of AD dementia that reflects the principles of TCM syndrome differentiation and treatment according to the time based on the discovery of the evolution of AD syndromes.However,the mechanism of action of this sequential therapy is not clear.In this study,by simulating the clinical "treatment according to time" model,different month-old APPswe/PS1 ΔE9(APP/PS1)double transgenic mice were given to tonifying kidney formula,resolve phlegm formula and detoxication formula,and the learning and memory of AD transgenic mice at different periods were tested Behavior and changes in the target targets of nuclear repressor element silencing transcription factor(REST),amyloid-β(Aβ)oligomer,phosphorylated tau(P-tau),Bcl-2 Associated protein X(Bax)and B cell lymphoma-2(Bcl-2)in the brain The effectiveness of sequential therapy in the treatment of AD and its neuroprotective mechanism provide a scientific basis for clinical sequential treatment of AD.Method1.Grouping:(1)Simulated early AD:3-month-old male APP/PS1 double transgenic mice were randomly divided into 12 groups,namely the low,medium and high dose group of tonifying kidney formula,and the low,medium and high dose group of resolve phlegm formula.detoxication formula low-,medium-and high-dose group,donepezil group.Normal wild type C57/BL6J was used as the normal control group,with 15 animals in each group.Donepezil group was given intragastric administration of donepezil hydrochloride(0.92 mg/kg/d),tonifying kidney formula high-dose group was given tonifying kidney formula(2.4 g/kg/d),and tonifying kidney formula medium-dose group was given tonifying kidney formula(1.2 g/kg/d).)Gavage,tonifying kidney formula low-dose group,tonifying kidney formula(0.6 g/kg/d)gavage,resolve phlegm formula high-dose group,resolve phlegm formula(3.0 g/kg/d)gavage,resolve phlegm formula medium-dose group,resolve phlegm formula(1.5 g/kg/d)intragastric,resolve phlegm formula low-dose group,resolve phlegm formula(0.75 g/kg/d)intragastric,detoxication formula high-dose group,detoxication formula(2.4 g/kg/d)Gavage and detoxication formula mid-dose group give detoxication formula(1.2 g/kg/d)gavage,detoxication formula low-dose group give detoxication formula(0.6 g/kg/d)gavage.The normal control group and the model group were gavaged with equal volume of 0.5%CMC.It is administered once a day with a volume of 0.1ml/10g for 3 months.(2)Mid-term AD simulation:A 3-month-old male APP/PS1 double-transgenic mouse was used to determine the superior dose group of each Chinese medicine group according to the experimental results,and they were randomly divided into tonifying kidney formula Group,resolve phlegm formula Group and detoxication formula Group,sequential therapy group,donepezil group.Normal wild type C57/BL6J was used as the normal control group,with 15 animals in each group.The donepezil group was given intragastric administration of donepezil hydrochloride(0.92 mg/kg/d),the tonifying kidney formula group was given tonifying kidney formula(1.2 g/kg/d),and the resolve phlegm formula group was given resolve phlegm formula(1.5 g/kg/d).Stomach,detoxication formula group was given intragastric administration of detoxication formula(1.2 g/kg/d),normal control group and model group were given intragastric administration of 0.5%CMC for 6 months,and sequential therapy group was given tonifying kidney formula(1.2 g/kg/d)gavage for 3 months,then resolve phlegm formula(1.5 g/kg/d)was gavaged for 3 months(3)Simulated advanced AD:3-month-old male APP/PS1 double transgenic mice were randomly divided into tonifying kidney formula Group,resolve phlegm formula Group,detoxication formula Group,Sequential Therapy Group,and Donepezil Group.Normal wild type C57/BL6J was used as the normal control group,with 15 animals in each group.The donepezil group was given intragastric administration of donepezil hydrochloride(0.92 mg/kg/d),the tonifying kidney formula group was given tonifying kidney formula(1.2 g/kg/d),and the resolve phlegm formula group was given resolve phlegm formula(1.5 g/kg/d).Stomach and detoxication formula group were given intragastric administration of detoxication formula(1.2 g/kg/d),normal control group and model group were intragastrically fed with 0.5%CMC for 9 months,and sequential therapy group was given tonifying kidney formula(1.2 g/kg/d)gavage for 3 months,then give resolve phlegm formula(1.5 g/kg/d)for 3 months,then detoxication formula(1.2 g/kg/d)for 3 months.2.Testing technology and methods:(1)Using Morris water maze and jumping platform detection sequential scheme to observe the learning and memory behavior changes of APP/PS1 double transgenic mice simulating AD early,middle and late onset.(2)Western blot and immunohistochemistry were used to detect the expression of Aβoligomer,P-tau,Bax,Bcl-2 protein and REST nuclear protein in mouse hippocampus.Result1.Effects of sequential therapy on spatial learning and memory ability of APP/PS1 mice simulating early onset of AD.(1)Swimming distance:on the 4th day of Morris water maze navigation experiment,compared with the model group,the swimming distance of the high and medium dose tonifying kidney formula group was significantly reduced,and the difference was statistically significant(P<0.05).On the 5th day,compared with the model group,the swimming distance of the medium dose,low dose of tonifying kidney formula group,and high dose groups of resolve phlegm formula group further decreased,the difference was statistically significant(P<0.05).(2)Escape incubation period:on the 5th day of the navigation experiment of water maze,compared with the model group,the escape incubation period of the high,medium and low dose of tonifying kidney formula group and the medium dose resolve phlegm formula group group were significantly reduced,and the difference was statistically significant(P<0.05)(3)Target quadrant residence time:in the water maze space exploration experiment,compared with the model group,the residence time in the target quadrant of the middle and low dose groups of tonifying kidney formula group increased significantly,and the residence time in the target quadrant of the middle dose group of resolve phlegm formula group also increased significantly,and the difference was statistically significant(P<0.05).(4)Number of platform errors:after 24 hours of platform training in mice,compared with the model group,the number of errors in the high-dose and low-dose tonifying kidney formula group was significantly reduced(P<0.05)2.Effects of sequential therapy on Aβ oligomers,P-tau,apoptosis-related proteins,and REST nucleoproteins in the hippocampus of APP/PS1 mice that simulated the early onset of AD(1)Western blot detection the expression of Aβ oligomers in hippocampus:compared with the model group,the expression of Aβ oligomers in the high-dose and medium-dose groups of tonifying kidney formula group and high-dose and medium-dose groups of resolve phlegm formula group were significantly reduced,with statistically significant differences(P<0.05).Immunohistochemical detection the Aβ oligomers of cortical and hippocampal:compared with the model group,the integrated optical density value(IOD)of Aβ oligomers in the high-dose and medium-dose groups of tonifying kidney formula group and the low-dose group of tonifying kidney formula group was significantly lower than that in the model group,with statistically significant differences(P<0.05).(2)Western blot detection the expression of P-tau protein:compared with the model group,P-tau protein expression was significantly reduced in the medium dose group of tonifying kidney formula group and the high-dose group of detoxication formula group,with statistically significant differences(P<0.05)(3)Ratio of bcl-2/bax detected by Western blot:compared with the model group,the ratio of bcl-2/bax in the low-dose group with tonifying kidney formula group was significantly higher than that in the model group,and the difference was statistically significant(P<0.05)(4)Westernblot detection the expression of REST nucleoprotein:compared with the model group,the expression of REST nucleoprotein increased significantly in the medium dose group of tonifying kidney formula group and the low dose group of resolve phlegm formula group(P<0.05),but there was no statistically significant difference in the expression of REST protein between the groups by immunohistochemical detection(P>0.05).3.Effect of sequential therapy on learning and memory ability of APP/PS1 mice simulating AD mid-term disease.(1)Swimming distance:on the 5th day of Morris water maze navigation experiment,the swimming distance of the sequential Therapy group was significantly reduced compared with that of the model group(P<0.05).(2)Escape incubation period:on the 4th day of the water maze navigation experiment,the escape incubation period of the mice in the resolve phlegm formula group was significantly reduced compared with that in the model group(P<0.05).On the 5th day,compared with the model group,the evasive incubation period of the mice in the resolve phlegm formula group was reduced,and the difference was statistically significant(P<0.05).(3)Target quadrant residence time:in the water maze space exploration experiment,compared with the model group,the residence time of resolve phlegm formula group increased significantly,and the difference was statistically significant(P<0.05).4.Effects of sequential therapy on Aβ oligomers,P-tau,apoptosis-related proteins,and REST nucleoproteins in the hippocampus of APP/PS1 mice that simulated the onset of mid-AD.(1)Westernblot detection the expression of Aβ oligomers:compared with the model group,the expression of Aβ oligomers was significantly decreased in the tonifying kidney formula group and resolve phlegm formula group,and the difference was statistically significant(P<0.05).(2)Western blot detection the expression of P-tau protein:there was no statistically significant difference in the ratios of P-tau/actin and P-tau/T-tau between groups(P>0.05).(3)Western blot detection the expression of apoptosis-related proteins:there was a statistical difference between the normal group and the model group(P<0.05),but there was no statistical difference between the treatment group and the model group(P>0.05).(4)Western blot detection the expression of REST nucleoprotein:the results showed no difference between each group(P>0.05).5.Effect of sequential therapy on learning and memory ability of APP/PS1 mice that simulated the late onset AD.(1)Swimming distance:on day 2,day 3 and day 5 of the navigation in the water maze,the swimming distance of mice in the model group increased compared with that in the normal group,with statistically significant difference(P<0.05),but there was no statistically significant difference between the model group and the drug administration group(P>0.05).(2)Escape incubation period:on day 2,day 3 and day 5 of the water maze navigation experiment,the escape incubation period of mice in the model group increased compared with that in the normal group,with statistically significant differences(P<0.05),but there was no statistically significant difference between the model group and the drug administration group(P>0.05)(3)Target quadrant residence time:in the water maze space exploration experiment,the results showed that there was no statistical difference in the target quadrant residence time between each group(P>0.05)6.Effects of sequential therapy on Aβ oligomers,P-tau,apoptosis-related proteins,and REST nucleoproteins in the hippocampus of APP/PS1 mice that simulated the late onset AD(1)Westernblot detection the expression of Aβ oligomers:compared with the model group,the expression of Aβ oligomers in the tonifying kidney formula group,resolve phlegm formula group,detoxication formula group and sequential Therapy group has decreased,but the difference was not statistically significant(P>0.05).(2)Western blot detection the expression of P-tau protein:the results showed that there was no statistically significant difference in the ratio of P-tau/actin between groups(P>0.05).(3)Western blot detection the expression of REST nucleoprotein:the results showed no statistical difference between each group(P>0.05)Conclusion1.Sequential therapy can improve the learning and memory ability of mice simulating the early stage of AD,reduce the deposition of Aβ oligomer,decrease the expression of P-Tau protein,and reduce the expression of apoptosis-related protein,considering that the mechanism may be related to the increased expression of REST nuclear protein.2.Sequential therapy can improve the learning and memory ability of mice simulating the middle stage of AD3.Resolve phlegm formula can improve the learning and memory ability of mice simulating the early stage of AD,reduce the deposition of Aβ oligomer and increase the REST nuclear protein.4.Resolve phlegm formula can improve the learning and memory ability and reduce the deposition of Aβ oligomer in mice simulating the middle stage of AD. |
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